1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA

In order to establish the Diels–Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4‐triazine‐modified 2′‐deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4‐triazine group was attached at the 5‐position of 2′‐deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2′‐deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN‐modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4‐triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5‐tetrazine for postsynthetic labeling by the Diels–Alder reaction with inverse electron demand.     

2′‐Deoxyribonucleoside Phosphoramidate Triphosphate Analogues as Alternative Substrates for E. coli Polymerase III

Thermostable bacterial polymerases like Taq, Therminator and Vent exo^? are able to perform DNA synthesis by using modified DNA precursors, a property that is exploited in several therapeutic and biotechnological applications. Viral polymerases are also known to accept modified substrates, and this has proven crucial in the development of antiviral therapies. However, non‐thermostable polymerases of bacterial origin, or engineered variants, that have similar substrate tolerance and could be used for synthetic biology purposes remain to be identified. We have identified the α subunit of Escherichia coli polymerase III (Pol III α) as a bacterial polymerase that is able to recognise and process as substrates several pyrophosphate‐modified dATP analogues in place of its natural substrate dATP for template‐directed DNA synthesis. A number of dATP analogues featuring a modified pyrophosphate group were able to serve as substrates during enzymatic DNA synthesis by Pol III α. Features such as the presence of potentially chelating chemical groups and the size and spatial flexibility of the chemical structure seem to be of major importance for the modified leaving group to play its role during the enzymatic reaction. In addition, we could establish that if the pyrophosphate group is altered, deoxynucleotide incorporation proceeds with an efficiency varying with the nature of the nucleobase. Our results represent a great step towards the achievement of a system of artificial DNA synthesis hosted by E. coli and involving the use of altered nucleotide precursors for nucleic acid synthesis.     

Screening the structural and functional properties of bicyclo-DNA: bc(ox)-DNA

The synthesis of two novel pyrimidine bicyclonucleosides (bc(ox)-nucleosides) has been accomplished. These bicyclonucleosides each carry a lipophilic benzyloxime substituent on the carbocyclic ring and show improved conformational similarity to 2'-deoxyribonucleosides as shown by their X-ray structures. The thymine-containing bc(ox)-nucleoside was converted into the corresponding phosphoramidite building block and incorporated into oligodeoxyribonucleotides by standard phosphoramidite chemistry. T(m) data with complementary RNA and DNA were measured and compared to corresponding cases of natural and unfunctionalized bc-DNA. It was found that single incorporations of bc(ox) residues destabilize duplexes by roughly 5 degrees C per modification. The destabilization was found to be due to the oxime substituent and not to the bicyclic scaffold itself. No significant alteration of the base-pairing selectivity as a function of the modification was observed. With RNA (but not with DNA) as a complement the relative thermal destabilization of bc(ox)-oligothymidylates was gradually reduced and converted into a stabilizing interaction with increasing numbers of consecutive modifications. While no cellular uptake of bc(ox)-oligonucleotides into HeLa cells occurred without transfecting agents, a significant increase in the transfection rate relative to unmodified DNA was observed in complexation with lipofectamine.     

Recombinase‐Based Isothermal Amplification of Nucleic Acids with Self‐Avoiding Molecular Recognition Systems (SAMRS)

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single‐stranded primers into the duplex DNA product; these are then extended using a strand‐displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base‐pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self‐avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS‐RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.     

Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7‐Substituted 7‐Deazaguanine Nucleobases

Previous studies of polymerase synthesis of base‐modified DNAs and their cleavage by restriction enzymes have mostly related only to 5‐substituted pyrimidine and 7‐substituted 7‐deazaadenine nucleotides. Here we report the synthesis of a series of 7‐substituted 7‐deazaguanine 2′‐deoxyribonucleoside 5′‐O‐triphosphates (dG^RTPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dG^RTPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7‐substituted 7‐deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage.     

Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases

C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo^? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis.     

Highly Efficient Quencher‐Free Molecular Beacon Systems Containing 2‐Ethynyldibenzofuran‐ and 2‐Ethynyldibenzothiophene‐Labeled 2′‐Deoxyuridine Units

We have prepared two fluorescent DNA probes—U^DBF and U^DBT, containing 2‐ethynyldibenzofuran and 2‐ethynyldibenzothiophene moieties, respectively, covalently attached to the base dU—and incorporated them in the central positions of oligodeoxynucleotides (ODNs) so as to develop new types of quencher‐free linear beacon probes and investigate the effect of functionalization of the fluorene scaffold on the photophysical properties of the fluorescent ODNs. The ODNs containing adenine flanking bases (FBs) displayed a selective fluorescence “turn‐off” response to mismatched targets with guanine bases; this suggests that these probes could be used as base‐discriminating fluorescent nucleotides. On the other hand, we observed a “turn‐on” response to matched targets when the U^DBF and U^DBT units of ODNs containing pyrimidine‐based FBs were positioned opposite the four natural nucleobases. In particular, an ODN incorporating U^DBT and cytosine FBs has potential use in single‐nucleotide polymorphism typing.     

Mechanism of Error‐Free Bypass of the Environmental Carcinogen N‐(2′‐Deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone Adduct by Human DNA Polymerase η

The environmental pollutant 3‐nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8‐dG‐ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error‐free manner by the human Y‐family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol?DNA?dCTP complex between a C8‐dG‐ABA‐containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error‐free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra‐nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson–Crick pair with the incoming dCTP.     

O6‐Alkylguanine‐DNA Alkyltransferase‐Mediated Repair of O4‐Alkylated 2′‐Deoxyuridines

O⁶‐Alkylguanine‐DNA alkyltransferases (AGTs) are responsible for the removal of O⁶‐alkyl 2′‐deoxyguanosine (dG) and O⁴‐alkyl thymidine (dT) adducts from the genome. Unlike the E. coli OGT (O⁶‐alkylguanine‐DNA‐alkyltransferase) protein, which can repair a range of O⁴‐alkyl dT lesions, human AGT (hAGT) only removes methyl groups poorly. To uncover the influence of the C5 methyl group of dT on AGT repair, oligonucleotides containing O⁴‐alkyl 2′‐deoxyuridines (dU) were prepared. The ability of E. coli AGTs (Ada‐C and OGT), human AGT, and an OGT/hAGT chimera to remove O⁴‐methyl and larger adducts (4‐hydroxybutyl and 7‐hydroxyheptyl) from dU were examined and compared to those relating to the corresponding dT species. The absence of the C5 methyl group resulted in an increase in repair observed for the O⁴‐methyl adducts by hAGT and the chimera. The chimera was proficient at repairing larger adducts at the O⁴ atom of dU. There was no observed correlation between the binding affinities of the AGT homologues to adduct‐containing oligonucleotides and the amounts of repair measured.     

Synthesis and Characterization of Amphiphilic Gd(III) Complexes: Gd‐DTPA‐BA

Magnetic resonance imaging (MRI) is widely used to identify different diseases. MRI contrast agents, used to enhance the MRI signal, have been studied extensively for precise diagnosis. Based on the advantages of macromolecular MRI contrast agents of higher contrast imaging ability and a longer cycle time, this article modified the most common micromolecular contrast agent (Gd‐diethylene triamine pentaacetic acid [DTPA]). 2 long saturated aliphatic chains were attached to both sides of DTPA. DTPA derivatives with 12, 14, and 16 carbon lengths were synthesized and chelated to Gd³⁺. 3 amphiphilic MRI contrast agents were obtained and their structures were characterized using mass spectrometry, ¹H NMR, and Fourier transform infrared. Furthermore, the surface tension of the compounds was measured, and liposomes were prepared by mixing the synthesized amphiphilic molecules with egg lecithin and cholesterol. The assembly behavior of the liposomes was studied using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements. TEM showed that the liposomes possessed bilayer vesicle structures. The liposome size distribution determined by DLS was from 10 to 1000 nm, and as the aliphatic chain length increased, the polydispersity index (PDI) and zeta potential increased. No obvious changes in the PDI and zeta potential of the liposomes were observed after 5 days at room temperature, suggesting that they possess good stability.     

Biochemical Characterization of a Uranyl Ion‐Specific DNAzyme

Uranyl ion‐specific DNAzyme : A DNAzyme (lower strand) cleaves the substrate (upper strand) in the presence of the uranyl ion. The enzyme folds into a bulged three‐way‐junction structure with catalytically important nucleotides residing in the bulge. A highly conserved G?A mismatch is also crucial for the enzyme's activity.

     

Synthesis and Characterization of Energetic 1,2‐Bis(Oxyamino)Ethane Salts

The synthesis, characterization, theoretical calculations, and safety studies of energetic salts based on 1,2‐bis(oxyamino)ethane, (H₂N O CH₂ CH₂ O NH₂), were carried out. The salts were characterized by vibrational (infrared, Raman), multinuclear NMR studies (¹H, ¹³C), differential scanning calorimetry (DSC), elemental analysis, and initial safety testing (impact and friction sensitivity). Single crystal X‐ray diffraction studies were carried out on the mono‐perchlorate and the double nitrate salts, revealing the expected structures.     

Protonation of Nucleobases in Single‐ and Double‐Stranded DNA

Single‐stranded model oligodeoxyribonucleotides, each containing a single protonatable base—cytosine, adenine, guanine, or 5‐methylcytosine—centrally located in a background of non‐protonatable thymine residues, were acid‐titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5‐methylcytosine base, were comparatively evaluated in single‐stranded and double‐stranded form, by UV spectroscopy and high‐field NMR. The N³ protonation of the 5‐methylcytosine moiety in the double‐stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single‐stranded case. The central guanine:5‐methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O² of the 5‐methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5‐methylcytosine sites in double‐stranded DNA might be protonated to a significant extent under physiological conditions.